A primary aim of this work is to extend our understanding of the relationship between the mechanism of nucleoside triphosphate (NTP) hydrolysis and the mechanical properties of muscle fibers with an emphasis upon determining which step or steps of the nucleotide triphosphate hydrolysis mechanism limits the maximum shortening velocity. Investigators have shown that a series of nucleoside triphosphates (NTPS) differ widely in their ability to support shortening and motility as measured in skinned muscle fibers (in collaboration with Drs. Roger Cooke and Ed Pate) and using the `in vitro' motility assay. In the first two years of this proposal the investigators plan to complete the study of the mechanism of hydrolysis of a series of nucleoside triphosphates by actomyosin using stopped-flow fluorescence, light scattering, and rapid chemical quench method. From this work they plan to determine for which steps of the hydrolysis mechanism changes in rate constants alter the mechanical and motile properties of muscle. To accomplish this the investigators will make a detailed comparison of the rate constants of the kinetic mechanism of the actomyosin NTP hydrolysis mechanism with the mechanical properties of muscle fibers and shortening velocity measured with the in vitro motility assay. A parallel approach is to determine the differences in amino acid sequence that are responsible for the differences in enzymatic and contractile activities of different myosins. A seven percent difference in the amino acid sequence between alpha and beta cardiac myosin primarily grouped in four clusters is responsible for the difference in mechanical and enzymatic properties of cardiac myosin isozymes. A similar pattern of more extensive sequence changes produces a three fold difference in the rates of actin activated ATP hydrolysis, a five fold difference in shortening velocity, and a ten fold difference in rate of ADP dissociation from acto-S1 between cardiac and fast skeletal myosin. Recent data (Uyeda et al. 1994) indicate that the rate of actin-activated ATP hydrolysis is dependent upon the sequence of amino acids 624-638, which are located in the junction between the 25 and 50 kDA regions of the myosin molecule. Investigators plan to use a combination of recombinant expression of chimeric myosins (chimerzymes), steady state kinetics, pre-steady state kinetics, and motility assay measurements to determine which amino acid changes are responsible for the observed differences in the kinetic mechanism and functional differences. Understanding which amino acids alter the rate and equilibrium constants of the hydrolysis mechanism and shortening velocity in muscle will provide a better understanding of the molecular mechanism of muscle contraction.